Received October 3, 2017; Revision received November 27, 2017
The view of the cell nucleus as a crowded system of colloid particles
and that chromosomes are giant self-avoiding polymers is stimulating
rapid advances in our understanding of its structure and activities,
thanks to concepts and experimental methods from colloid, polymer, soft
matter, and nano sciences and to increased computational power for
simulating macromolecules and polymers. This review summarizes current
understanding of some characteristics of the molecular environment in
the nucleus, of how intranuclear compartments are formed, and of how
the genome is highly but precisely compacted, and underlines the
crucial, subtle, and sometimes unintuitive effects on structures and
reactions of entropic forces caused by the high concentration of
macromolecules in the nucleus.
KEY WORDS: cell nucleus, macromolecular crowding, confinement,
entropic forces, phase separation, chromosomes

DOI: 10.1134/S0006297918040041

“The study of the colloidal state is … of special
importance since the vast majority, if not all, of the substances of
which protoplasm is made up have very large and complicated particles
and therefore must give colloidal solutions” [1]. This insight of Alexander Oparin, published close
to 100 years ago, was neglected until recently. Now, its implications
are leading to rapid progress in our understanding of
“protoplasm” and particularly of the cell nucleus, based on
the properties of colloids and their interactions with polymers like
chromosomes and the experimental and simulation methods that have been
developed to study them.

Most of us who work with nuclei or subcellular systems believe, and
sometimes state in publications, that our experimental conditions are
“physiological” and that our macromolecules are influenced
only by van der Waals, electrostatic, hydrogen bonding, and hydrophobic
forces [2]. Until recently, most biologists did not
realize the significance of the discovery by Asakura and Oosawa in 1958
that “An attractive force appears between particles suspended in
solutions of macromolecules … that becomes stronger in solutions
of chain macromolecules or of macromolecules of dissymmetrical shape
than in solutions of rigid spherical macromolecules … and can
have a remarkable influence on the state of suspended particles”
[3]. These strong and subtle attractive forces,
termed entropic or depletion forces, are negligible in the dilute
systems of macromolecules that we usually use, but they become very
important at high concentrations when macromolecules are in close
proximity. This parameter, termed macromolecular crowding, is a common
feature of all parts of biological cells, the cytoplasm [4, 5], membranes [6], and mitochondria [7], and of
intercellular spaces in tissues [8] and is an
essential but frequently overlooked feature of
“physiological” conditions that should be reproduced in
experiments. This review describes some characteristics of the
molecular environment in the nucleus and underlines how insights and
simulations from colloid, polymer, soft matter, and nano sciences are
contributing to answering the far-sighted question of Francisco Iborra:
“Can visco-elastic phase separation, macromolecular crowding and
colloidal physics explain nuclear organization?” [9] and to understanding the structures and properties
of the nucleus and the genome.

EFFECTS OF CROWDING ON MACROMOLECULES

Entropic attractive forces. Entropic forces between
macromolecules arise in a concentrated mixture because when the
molecules are close enough, others cannot enter the regions between
them and the forces that they exert because of random (Brownian)
movement are no longer symmetrical (Fig. 1). By the
same principle, entropic forces favor the localization of particles on
a surface [10] and helical coiling of linear
molecules [11].

Fig. 1. Attractive entropic forces. a) In a concentrated mixture,
particles or macromolecules are so close that others cannot enter the
shaded regions between them and therefore the forces (arrows) caused by
random Brownian movement of the black macromolecules are no longer
symmetrical. The second law of thermodynamics (states with maximum
entropy are favored) is then satisfied because the volumes around the
large macromolecules that are not accessible to the black molecules
(dashed lines) overlap in the shaded region, increasing the volume
available to them. b) Self-association of spherical particles driven by
entropic forces as the concentration of smaller particles (not seen) in
a mixture increases from (1) to (3) (reproduced from [12] with permission of the Royal Society). c, d)
Entropic self-association of nuclear macromolecules: c) a 3 kb mRNA
incubated with 1% (control) or 20% PEG (35 kDa) (reproduced from
[13] with permission of the American Society for
Biochemistry and Molecular Biology); d) polynucleosomes (DNA length
∼ 150 kb) labeled with YOYO-1 incubated without or with 12.5% PEG
(8 kDa) and spread on a surface (reproduced from [14] with permission of Springer).

Crowded systems have a second particularity: the effective volume of a
macromolecule from which others are excluded exceeds its intrinsic
volume, and for a spherical macromolecule this effective volume is 8
times its intrinsic volume. Thus, even when macromolecules appear to
occupy only a small part of a total volume, there may remain no space
for others of the same size, resulting in a significant increase of
their thermodynamic activity.

In studies of effects of crowding in vitro, volume-occupying
synthetic polymers such as poly(ethylene glycol) (PEG), dextran, and
Ficoll are widely used as “crowding agents”, but it is
important to recognize that these do not reproduce perfectly the
effects of the complex crowded environments in vivo, as
discussed in the last section of this review.

Effects on proteins. Crowding has strong effects on the folding,
conformation, stability, interactions, and biological activities of
proteins that have been described in several comprehensive reviews [15-18]. The few studies in
vivo suggest that these effects depend on the protein and the local
environment [19]. Apparently the only nuclear
protein that has been studied is histone H1, whose intrinsically
disordered C-terminus is converted to a molten globule state in crowded
conditions [20].

Effects on DNA and RNA conformation. Numerous changes of DNA
conformation in crowded conditions have been reported, implying that
conformations in living cells may be different from those in in
vitro, but their implications are not yet clear because complex
interactions occur between ionic effects and crowding [21]. A model DNA duplex
(5′-TAGGTTATAA-3′/5′-TTATAACCTA-3′) shows a
7°C decrease of melting temperature when crowded in 20% PEG (200
Da) [22]. Triplex formation by poly(dA)-poly(dT)
in vitro is promoted strongly in crowded conditions [23], and an intramolecular i-motif structure
of triplet-repeat DNA oligomers is induced by crowding [24, 25]. The formation of
G-quadruplexes in double-stranded DNA is favored by crowding [26], but its influence on the formation of telomeric
G-quadruplexes in vivo is not yet clear. Folding of RNA is also
promoted by crowding, with dramatic stabilization of folded states [27, 28].

Entropic interactions can be highly specific. Entropic forces are
highly sensitive to the local shape of interacting surfaces, and
therefore show selectivity of a “lock and key” type
(Fig. 2) [29]. Their
ability to produce torque could be significant when a macromolecule has
to adopt a particular orientation for its function [29]. Because of their ability to organize particles
and molecules precisely, entropic interactions are studied widely for
the design of nanostructures [30, 31].

Fig. 2. Entropic interactions are highly sensitive to shape. a)
Left: a “key” molecule that fits precisely into a cavity on
another molecule feels a strong entropic attraction, because other
molecules cannot enter the interface. If the fit is not precise
(right), other molecules can enter the interface and weaken the
entropic attraction. b) Snapshots that show this selectivity during
self-assembly of nanoparticles (reproduced from [31] with permission of Nature Publishing
Group).

CROWDING AND THE STRUCTURE OF THE NUCLEUS

Concentration of macromolecules in the nucleus. Different methods
confirm that nuclei contain macromolecules at a global concentration of
at least 100-200 mg dry mass/ml (Fig. 3).
This high concentration is maintained by confining the contents of the
nucleus within the mechanically strong but somewhat elastic nuclear
envelope [32] (Fig. 4b).

Fig. 3. a) Concentrations of macromolecules in the nucleus and
its internal compartments; references are from [33]. b) Snapshot from a simulation of a similar
macromolecular environment, the cytoplasm of E. coli, where the
global concentration is somewhat higher than that in the nucleus but
similar to that in the nucleolus (reproduced from [34], image kindly provided by Adrian
Elcock).

Fig. 4. Formation of compartments in the nucleus by crowding: a)
the upper row shows phase contrast images of isolated nuclei, and the
center and lower rows show PML or Cajal bodies visualized using
fluorescent antibodies to their major protein. Left column, nuclei in
normal buffer; center column, disassembly of compartments when nuclei
expand after incubation in low ionic strength buffer; right column,
reassembly of compartments after further incubation with addition of
12% PEG (8 kDa); b) the contents of the nucleus are confined and
compressed by the nuclear lamina, visualized here by immunolabeling of
lamin A. The lamina of isolated nuclei (left) contracts after DNA is
digested with restriction enzymes and chromatin is removed by
electroelution (right) [R. Hancock, unpublished
data].

Compartments in the nucleus. At least 20 types of compartments
have been visualized by optical microscopy within nuclei under
different conditions, of which the most ubiquitous are nucleoli, Cajal
and PML bodies, splicing speckles, gems, and sites
(“factories”) where RNA transcription or DNA replication
occur [35]. These compartments have characteristic
properties: absence of a surrounding membrane, mobility within the
nucleus, and dynamic exchange of their components with the surrounding
nuclear space.

A crucial clue to the formation of these compartments was provided by
Oksana Dudnik, Olga Zatsepina, and Yuri Chentsov who discovered by
optical microscopy that nucleoli disassemble almost completely when
living cells are incubated in a hypotonic medium, and they reform when
the normal medium is restored [36]. Subsequently,
it was found that Cajal and PML bodies [37], DNA
replication foci [38], and interactions of
chromatin that regulate gene expression [39] (see
further Fig. 7) also disassemble in hypotonic
conditions. Isolated nuclei increase considerably in volume in
hypotonic media, suggesting that these effects are caused by a decrease
of the concentration of the macromolecular components of these
compartments. This model is supported by the observation that they
reassemble when the nuclear volume is restored by adding a
macromolecular crowding agent [37, 39] (Fig. 4; see further
Fig. 7).

Simulations of systems of concentrated macromolecules support the idea
that nuclear compartments are assembled from their component
macromolecules by crowding [40, 41] (Fig. 5a). Entropic
forces favor the localization of particles on a surface [10], and simulations explain why nuclear compartments
are frequently associated with the surface of chromosomes [41] (Fig. 5b). Nuclear
compartments therefore appear to be the results of purely
physicochemical interactions in the crowded nucleoplasm, suggesting
that the continuing search for specific functions of compartments such
as PML and Cajal bodies may not be realistic.

Fig. 5. Simulations by molecular dynamics to understand the
formation of compartments in the nucleus and their tendency to
associate with chromosomes. a) Clusters of spherical particles (red)
are predicted to form when the volume fraction (φc) of
crowding particles (blue) increases (reproduced from [40] with permission from Elsevier). b) In the
presence of a polymer chain (red), clusters (gray) are predicted to
form and then associate with the polymer when the particle density is
high and particle–polymer interactions are moderately weak
(above), while for strong interactions and lower particle densities the
clusters grow in contact with the polymer (below) (reproduced from [41] with permission of the Royal Society of
Chemistry).

Nuclear compartments as liquid drops produced by phase
separation. When attraction due to entropic and other forces is
strong enough in a mixture of particles or particles and polymers, the
mixture may separate and form discrete phases, and crowding stimulates
phase separation by increasing the effective concentrations in a
mixture [42, 43]; this is
seen in the simulations in Fig. 5 when
clusters of particles are formed. The idea that compartments in the
nucleus are formed by phase separation (of which coacervation is a
particular type) was proposed in quite early studies; Albert
Frey-Wyssling wrote in 1938 that “the nucleus … may behave
like a liquid drop … nucleolus formation must be considered as
an accumulation of the karyolymph proteins … until a coacervate
droplet rich in proteins is formed” [44],
and in 1946 Lars Ehrenberg concluded that “the nucleolus is a
separated phase out of a saturated solution… Its globular shape
and the absence of discontinuities as well as the mode of deformation
of the persisting nucleolus by the spindle suggest that it is a
drop” [45]. Phase separation requires a
critical concentration of some component(s) [42,
46, 47] and is therefore
closely dependent on local level of crowding. The concept that
“the nucleus … may behave like a liquid drop” and
that “the nucleolus is a separated phase” with
“coacervate nature” has now become a major theme in cell
biology [46, 47].

CROWDING, CONFINEMENT, AND THE CONFORMATION OF THE GENOME

The dense crowding of ∼65 cm of genomic chromatin into a
∼6 μm diameter nucleus implies that there must be abundant
contacts between different regions of the genome. Earlier, at least
some contacts were believed to be stable and to result in the formation
of the loops seen by optical microscopy, for example [48].

Simulations show that entropic forces favor the formation of loops in a
self-avoiding polymer chain like a chromatin fiber [49-53]. In some models,
inter-chain contacts are proposed to be mediated through protein
“binders” [54], but most simulations
find that loops form spontaneously when a self-avoiding chain folds in
a confined space, without the need to postulate “binders”
[55-58]. Comprehensive
simulations have led to the Dynamic Loop model [51, 52] and show that the
formation of loops results solely from diffusional motion of a polymer,
without other long-range interactions. Introducing loops in polymer
chains strongly increases the probability of contacts between distant
regions compared to linear chains, and it also results in a multi-fold
higher repulsion between them due to entropic forces compared to linear
chains, reproducing the discrete form of chromosome territories in
vivo (Fig. 6).

Fig. 6. Simulations showing that in a confined volume,
chromosomes with larger portion of loops are separated more completely,
like chromosome territories in vivo. a) Linear polymer chains
have abundant contacts with others; the fraction of inter-chain
contacts is 15.4%. b, c) Increasing the portion of polymer organized in
loops (the number of loops) results in more and more confined
structures with fewer inter-chain interactions. For chains with an
average of 45 functional loops (b) the fraction of contacts is reduced
to 1.8% (reproduced from [51]).

The dynamic nature of loops was observed quite early [56], and only dynamic loops reproduce the chromatin
interaction frequencies observed by chromosome conformation capture [57]. Entropic forces and confinement are also
predicted to cause the formation of topologically associating domains
(TADS), regions of the genome where chromosomal interactions are
particularly frequent [58].

The formation by crowding of functional loops in chromatin in
vivo to bring a gene into contact with its remote regulatory
elements has been shown clearly by chromosome conformation capture (3C)
experiments on the β-globin gene domain of the mouse genome [39]. In nuclei in isotonic buffer the frequency of
contacts between upstream regulatory elements and the gene domain is
high, but these contacts are disrupted when nuclei expand in hypotonic
conditions. The contacts are re-established when the crowder PEG is
added to the expanded nuclei (Fig. 7).

Fig. 7. Crowding in nuclei drives the interaction between the
β-globin gene domain of the genome and its remote regulatory
elements. Chromosome conformation capture was performed on nuclei in
isotonic conditions (“nuclei”), expanded in hypotonic
buffer (“hypotonic nuclei”), or in hypotonic buffer with
addition of 12.5% PEG (8 kDa) (“recovery”). The
vertical scale shows the relative frequency of ligation of the fragment
harboring the 3′-insulator (colocalized with the 3′ DNase
I-hypersensitive site (3′HS)) with other fragments of the domain,
reflecting the frequency of contact between these regions. The map
(top) shows the β-globin genes, olfactory receptor genes, and HS;
0 kb corresponds to the start of the Hbb-y gene. The dark gray
rectangle shows the anchor fragment and the light gray rectangles
indicate test fragments (adapted from [39]).

EFFECTS OF CROWDING ON FUNCTIONS IN THE NUCLEUS

Transcription. Transcription of mRNAs in in vitro systems
is stimulated by crowding agents. For example, the production of mRNA
for Renilla luciferase in wheat germ extract is enhanced
∼4-fold in the presence of 20% Ficoll (70 kDa) (Fig. 8a); the stimulation by 40% Ficoll is smaller,
probably due to increased viscosity and slower dissociation of some
enzyme–substrate intermediates [59].
Crowding increases the rate of mRNA production significantly in
picolitre water-in-oil droplets containing a coacervated lysate of
E. coli [60]. A good example of sometimes
subtle effects of crowding is seen in a model of the sporadic pulses or
“bursts” of transcription that occur in vivo [61]; if polymerase molecules working along a gene
meet, entropic attraction between them causes them to form a stable
peloton or cluster that moves together along the gene and results in a
burst of transcription [62].

Fig. 8. Stimulation by crowding of reactions involved in
transcription and replication: a) effect of Ficoll (70 kDa) on
synthesis of mRNA for Renilla luciferase (Rluc) in an in
vitro transcription system (reproduced from [59]); b) effect of 8 kDa PEG (12%) on the
affinity of DNA polymerase I for DNA (reproduced from [64]).

DNA replication. Crowding stimulates several individual steps
related to DNA replication [63] including the
activity of DNA helicase, the formation of the T4 DNA polymerase and
other intermolecular complexes, and the affinity of DNA polymerase I
for DNA (Fig. 8b) [64].

In growing cells, the sites of DNA replication are seen as foci in
nuclei, which were earlier interpreted as replication
“factories” through which the DNA of multiple replicons
moves and is replicated. The observation that the number of foci
increases markedly when nuclei expand upon incubation of cells in
hypotonic medium [38], however, challenged this
model and showed that foci are in fact assemblies of smaller structures
that are probably associated reversibly by crowding effects like other
intranuclear compartments. Now, super-resolution microscopy in three
dimensions resolves several thousand replication foci per nucleus, each
corresponding to a single replicon [65].

Diffusion, signaling, and finding targets. In the
nucleus, macromolecules diffuse several-fold more slowly than in
aqueous solution and even more slowly within the nucleolus. Their
subdiffusion is anomalous, showing that their movement is hindered by
obstacles [66-68].
Surprisingly, simulations show that this increases the probability of
ﬁnding a nearby target and consequently facilitates the
propagation of signals [69]. Crowding has a
potentially important influence on signaling pathways, which frequently
include a cascade of steps where intermediates are phosphorylated at
two sites; since protein kinases diffuse slowly in a crowded
environment, after a first phosphorylation they are more likely to
rebind to intermediates and to phosphorylate them again, resulting in
more processive responses, which have been confirmed by experiments
in vitro [70].

Factors tht bind to and regulate activities of DNA find their targets
rapidly, although competing nonspecific binding sites are in very large
excess, and they are believed to search by nonspecific binding and then
sliding and hops and jumps [71]. The compact and
looped conformations of DNA in vivo imposed by crowding and
confinement facilitate jumping between DNA segments in close proximity,
and looping helps them to bypass other factors bound to DNA while
searching [72].

Crowding and the structure of nuclei and mitotic chromosomes in
vivo. Nuclei and chromosomes in vivo are expected to
feel strong crowding effects from the surrounding cytoplasm, which
contains diffusible macromolecules at ~130 mg/ml [73, 74]. They are usually
isolated in media without crowding agents to reproduce these
conditions, but which contain Mg2+ and/or other cations
at millimolar concentrations. However, nuclei and chromosomes are
perfectly stable in solutions that contain only a crowding
macromolecule to mimic the cytoplasmic environment, without cations
and/or polyamines (Fig. 9), raising the
question if the conditions generally used for their isolation are
really “physiological” and reproduce the situation in the
cell. This paradox is illustrated by the fact that chromatin incubated
with cations in vitro has a different conformation from that in
nuclei or chromosomes in vivo [75].
Together, these arguments support the hypothesis that the integrity of
nuclei and chromosomes in vivo is strongly influenced by
crowding from cytoplasmic macromolecules. Several other cases are known
where crowding can replace or vastly reduce a requirement for
Mg2+ to stabilize macromolecular assemblies, for example
ribosomes [76] and bacterial chromosomes [77], or to stimulate their reactions, for example
ribozymes [27] and recA protein [78]. The quantities of ions in the nucleus and
cytoplasm have been measured, but the concentrations of osmotically
active ions cannot be calculated because the volume, in which they are
dissolved, and the fractions bound to macromolecules are not known, and
it has been argued that most cations in the cell are bound to
macromolecules [79].

All known types of DNA genomes appear to require a crowded environment
for their structure and activities, and crowded and confined conditions
appear to have been fundamental for the evolution of life [80]. Bacterial cells are even more crowded than
eukaryotic nuclei, and entropic forces drive the separation of their
daughter genomes [81]. The intracellular milieu of
dinoflagellates is so crowded that the genome has a liquid crystal
conformation [82]. The strong and subtle effects
of crowding on structures and activities in the nucleus that are
discussed here suggest some plausible reasons why a crowded milieu was
essential for evolution: the conformations and interactions of
macromolecules and chromosomes are exquisitely sensitive to entropic
forces; in crowded conditions, the thermodynamic activity of
macromolecules is enhanced so that interactions proceed efficiently
with a smaller number of molecules; and molecular interactions under
entropic forces may be more readily reversible than those caused by
ionic forces, for example DNA condensed by PEG remains more flexible
and less compact than when it is condensed by electrostatic
interactions [83]. Crowded conditions could
provide a form of “metabolic buffering” to stabilize the
complex network of molecular interactions in the nucleus [66].

QUESTIONS FOR THE FUTURE

Measurement of crowding in vivo. Local levels of crowding
in the nucleus are expected to vary depending on the local
macromolecular environment. Sensors that respond to crowding are being
developed and should give useful information if they can be targeted to
specific regions [84-86].
In-cell NMR [19, 87, 88] has not yet been applied to examine proteins in
the nucleus.

Crowders to mimic the conditions in vivo. Common,
so-called inert crowders like PEG are not hard spherical particles like
those used for theoretical simulations. They have subtle effects on the
structure, stability, and functional activity of macromolecules that
cannot yet be predicted and can show weak attractive interactions that
destabilize protein complexes, in contrast to the stabilization
predicted by entropic effects [17, 89]. For example, when telomeric repeat DNA is
crowded by PEG, its conformation is not the same as that formed with
Ficoll or in an extract of Xenopus laevis eggs, suggesting that
in this case PEG does not correctly reproduce the effects of the
crowded milieu in vivo [90]. Some studies
go so far as to suggest that commonly used crowders do not yield
physiologically relevant information [89, 91].

Crowding and the movement of macromolecules. A novel aspect of
crowding has been revealed by computational methods [92]; local heterogeneity of the level of crowding is
predicted to cause directional movement of other molecules. This effect
has potentially important implications for understanding movements of
molecules in vivo, for example movements of chromatin [93] and of newly-synthesized mRNAs away from
chromatin [94].

“Lock and key” interactions. When considering the
mechanisms by which covalent modifications of proteins or DNA influence
their interactions with other macromolecules, the fact that local
shapes of molecular surfaces strongly influence entropic interactions
(Fig. 2) is often overlooked. For example, the
effects of phosphorylation of a protein are generally interpreted in
terms of modified hydrogen bonds or ionic interactions through the
phosphate group [95], but they could be the result
of a change of surface shape that is recognized and bound entropically
by a “key” motif on binding partners. Similarly,
methylation of DNA sequences causes a change of their conformation [96], which could be recognized by entropic
“keys”.

Crowding homeostasis. Structures and activities in the nucleus
are clearly highly dependent on and sensitive to the level of crowding,
implying that “crowding homeostasis” must be essential [97, 98]. Signaling pathways that
control the level of crowding by precisely equilibrating the exit of
messenger RNAs with the entry and exit of proteins through nuclear
pores must exist but have not yet been explored.